Thistlethwaite, I., Bull, F., Cui, C., Walker, P., Gao, S-S., Wang, L.,Song, Z., Masschelein, J., Lavigne, R., Crump, M., Race, P.,Simpson, T., & Willis, C. (2017). Elucidation of the relative andabsolute stereochemistry of the kalimantacin/batumin antibiotics.Chemical Science, 8(9), 6196-6201.https://doi.org/10.1039/c7sc01670k

Publisher's PDF, also known as Version of recordLicense (if available):CC BYLink to published version (if available):10.1039/c7sc01670k

Link to publication record in Explore Bristol ResearchPDF-document

University of Bristol - Explore Bristol ResearchGeneral rights

This document is made available in accordance with publisher policies. Please cite only thepublished version using the reference above. Full terms of use are available:http://www.bristol.ac.uk/red/research-policy/pure/user-guides/ebr-terms/

ChemicalScience

EDGE ARTICLE

Ope

n A

cces

s A

rtic

le. P

ublis

hed

on 1

1 Ju

ly 2

017.

Dow

nloa

ded

on 1

7/07

/201

7 14

:08:

41.

Thi

s ar

ticle

is li

cens

ed u

nder

a C

reat

ive

Com

mon

s A

ttrib

utio

n 3.

0 U

npor

ted

Lic

ence

.

View Article OnlineView Journal

Elucidation of th

aSchool of Chemistry, University of Bristol

E-mail: chris.willis@bristol.ac.ukbLaboratory of Gene Technology, KULeuven,cSchool of Biochemistry, University of Bristo

† Electronic supplementary information (materials and methods and additional tfull 1H and 13C NMR data for all new com

‡ Present address: Department of Chemist7AL, UK.

Cite this: DOI: 10.1039/c7sc01670k

Received 14th April 2017Accepted 2nd July 2017

DOI: 10.1039/c7sc01670k

rsc.li/chemical-science

This journal is © The Royal Society of

e relative and absolutestereochemistry of the kalimantacin/batuminantibiotics†

Iain R. G. Thistlethwaite,a Freya M. Bull,a Chengsen Cui, a Paul D. Walker, a

Shu-Shan Gao, a Luoyi Wang,a Zhongshu Song,a Joleen Masschelein,‡b

Rob Lavigne, b Matthew P. Crump, a Paul R. Race, c Thomas J. Simpson a

and Christine L. Willis *a

Kalimantacin A and batumin exhibit potent and selective antibiotic activity against Staphylococcus species

including MRSA. Both compounds are formed via a hybrid polyketide synthase/non-ribosomal peptide

synthetase (PKS-NRPS) biosynthetic pathway and from comparison of the gene clusters it is apparent that

batumin from Pseudomonas batumici and kalimantacin from P. fluorescens are the same compound. The

linear structure of this unsaturated acid was assigned by spectroscopic methods, but the relative and

absolute stereochemistry of the five stereocentres remained unknown. Herein we describe isolation of

kalimantacin A and two further metabolites 17,19-diol 2 and 27-descarbomyl hydroxyketone 3 from cultures

of P. fluorescens. Their absolute and relative stereochemistries are rigorously determined using

a multidisciplinary approach combining natural product degradation and fragment synthesis with

bioinformatics and NMR spectroscopy. Diol 2 has the 5R, 15S, 17S, 19R, 26R, 27R configuration and is the

immediate biosynthetic precursor of the bioactive kalimantacin A formed by oxidation of the 17-alcohol to

the ketone.

Introduction

Polyketides isolated from bacteria exhibit a range of importantbioactivities making them attractive leads for the developmentof therapeutics,1 for example mupirocin is used worldwide asa topical antibiotic.2 These compounds are efficiently assem-bled in the host microorganism via sophisticated multipleenzyme architectures known as modular polyketide synthases.The rational alteration of these biosynthetic pathways offersexciting opportunities to access novel bioactive agents.3

In the course of a screening program for novel antibiotics,the kalimantacins were isolated from cultures of Alcaligenes sp.YL-02632S and the major metabolite, kalimantacin A 1 wasshown to exhibit activity against Gram-positive bacteriaincluding MRSA (Scheme 1).4,5

, Cantock's Close, Bristol BS8 1TS, UK.

Leuven B-3001, Belgium

l, Bristol BS8 1TD, UK

ESI) available: Complete description ofables, gures and schemes includingpounds. See DOI: 10.1039/c7sc01670k

ry, University of Warwick, Coventry CV4

Chemistry 2017

A short time later the antibiotic batumin was isolated fromPseudomonas batumici and found to have the same molecularweight and spectroscopic properties as kalimantacin A.6,7

Batumin has been patented in the Ukraine and formulated asDiastaf and used to detect staphylococci by taking advantage ofits selectivity for these strains.8 Decreased biolm formationhas also been reported for the majority of S. aureus strainsinvestigated9 and it has been found to reduce nasal S. aureuscarriage.10 Peschel and co-workers have recently highlighted thepotential scope of narrow spectrum antibiotics in the control ofbacterial populations.11

In 2010 Lavigne and co-workers isolated a metabolite fromcultures of Pseudomonas uorescens BCCM_ID9359 which againhad spectroscopic properties in accord with the structure ofkalimantacin and batumin hence it was named kal/bat(Scheme 1). The kal/bat gene cluster was identied and char-acterized and shown to consist of 16 open reading framesencoding a hybrid modular polyketide synthase/non-ribosomalpeptide synthetase (PKS-NRPS) system.12,13 It has recently beenestablished that the biosynthetic gene clusters of kalimantacinfrom P. uorescens and batumin from P. batumici14,15 are iden-tical, conrming that they both produce the same naturalproduct based on a structural framework assembled on a linearunsaturated acid featuring ve stereocenters. The PKS-NRPS isa member of the trans-acyltransferase (AT) class of modularpolyketide synthases that lack integral AT-domains whereby

Chem. Sci.

Scheme 1 Proposed final stages of the biosynthesis of kalimantacin A.

Chemical Science Edge Article

Ope

n A

cces

s A

rtic

le. P

ublis

hed

on 1

1 Ju

ly 2

017.

Dow

nloa

ded

on 1

7/07

/201

7 14

:08:

41.

Thi

s ar

ticle

is li

cens

ed u

nder

a C

reat

ive

Com

mon

s A

ttrib

utio

n 3.

0 U

npor

ted

Lic

ence

.View Article Online

they gain AT and other tailoring activities in trans.16,17 This candramatically increase the diversity and complexity of thechemical transformations available to the pathway and offerssignicant potential for pathway engineering of newcompounds. New metabolites diol 2, with a 17-hydroxyl grouprather than carbonyl, and descarbamoyl compound 3 wereisolated by inactivation of the last two tailoring genes batF(carbamoyl transferase) and batM (oxidoreductase) of theP. uorescens gene cluster (Scheme 1).

The nal conversion by BatM was particularly interesting asit transformed the almost inactive precursor, diol 2, to thebioactive 17-ketone 1. This key step was hypothesized to beessential for bacterial physiology whereby production of theinactive precursor functions as a pro-drug to avoid self-toxicity.12,13 The mode of action of kal/bat was suggested to beinhibition of bacterial fatty acid biosynthesis, due to the pres-ence of a FabI isoform, namely BatG and the antibiotic has highspecic activity against staphylococci.13,18 Kalimantacin/batumin has potential value as a lead antibiotic, exhibitingselective and high specic activity against staphylococci (MICvalue of 0.064 mg mL�1) and is signicantly more active thanother antibiotics in commercial use (e.g. mupirocin MIC ca. 0.5mg mL�1).

The relative and absolute structures of kal/bat 1 and putativebiosynthetic intermediates 2 and 3 have remained elusive usingspectroscopic methods or via attempts to generate a crystallinederivative for X-ray studies. With 32 possible isomers it isimportant to determine the full structure of this antibiotic.Herein we report a multidisciplinary approach combiningnatural product isolation, chemical degradation and fragmentsynthesis with bioinformatics and extensive NMR studies todetermine the relative and absolute conguration of 1 and 2isolated from P. uorescens. For simplicity we use the namekalimantacin.

Scheme 2 Stereoselective reduction of kalimantacin A 1 and aceto-nide formation.

Results and discussion

Initially, preparative amounts of pure kalimantacin A 1 wereisolated from P. uorescens for degradation and derivatizationstudies. We found that higher titres were obtained by growingcultures in a modied L-medium rather than a tryptose brothsupplemented with sucrose and glycine as reported previ-ously.12 The natural product was readily puried, using eithernormal phase followed by reversed phase silica chromatography

Chem. Sci.

or HPLC to yield typically 50mg L�1 of kalimantacin A. A similarprotocol was used to grow the DbatF and DbatM mutants ofP. uorescens giving biosynthetic intermediates 2 and 3. Thespectral data of 1, 2 and 3 were in accord with those reportedpreviously12 and these compounds were found to be stable forseveral weeks at room temperature.

The rst structural investigation was the relative stereo-chemistry of the 17,19-diol in 2 isolated from the DbatMmutantof P. uorescens. Directed reductions19,20 of the b-hydroxyketonemoiety of kalimantacin A 1 gave 1,3-syn and 1,3-anti diols 4 and5 for comparison with diol 2 (Scheme 2). The optical rotation ofsynthetic anti diol 5, [a]D �10.0 and natural product 2, [a]D�10.4 correlated well and their 1H and 13C NMR spectra wereidentical. In contrast 1,3-syn diol 4 with [a]D �5.9 showedsignicant differences in the chemical shis of C-17 (dc 68.4for 2, dc 70.3 for 4) and C-19 (dc 66.3 for 2, dc 72.0 for 4) (Tables 1and 2, ESI†).

The assignment of the relative stereochemistry of the 1,3-synand 1,3-anti diols 4 and 5 was conrmed using the method of

This journal is © The Royal Society of Chemistry 2017

Scheme 3 Oxidative cleavage of diol 2.

Edge Article Chemical Science

Ope

n A

cces

s A

rtic

le. P

ublis

hed

on 1

1 Ju

ly 2

017.

Dow

nloa

ded

on 1

7/07

/201

7 14

:08:

41.

Thi

s ar

ticle

is li

cens

ed u

nder

a C

reat

ive

Com

mon

s A

ttrib

utio

n 3.

0 U

npor

ted

Lic

ence

.View Article Online

Rychnovsky and Evans21 from the characteristic quaternary andmethyl group 13C chemical shis of the corresponding aceto-nides 6 and 7 (Scheme 2). Similarly the natural diol 2 wasconverted to its acetonide and was the same by NMR to 7 con-rming the anti relationship in the 17,19 diol. Bioinformaticanalysis of active site motifs of the appropriate modular ketor-eductase (KR) domains predicts that the 17-alcohol should beintroduced with the “R” conguration on reduction of the b-keto–thiol ester assembly intermediate.12 Hence taking intoaccount the Cahn–Prelog–Ingold priority change upon furtherchain elongation, we made a preliminary assignment of theabsolute conguration for diol 2 as 17S, 19R.

Sequence analysis of the relevant KR domain suggested the Rconguration at C27 but this, as well as the relative stereo-chemistry of C26 and C27, remained to be proven.12 Thus, twofragment mimics, (2R,3R,20R)-diol 8 and (2S,3R,20R)-diol 9, ofkalimantacin A were prepared and varied in the conguration ofthe 2-methyl groups (see ESI for synthetic details†). Their NMRspectra were compared with those of the descarbamoyl metabo-lite 3 isolated from theDbatFmutant of P. uorescens and the bestt was with (2R,3R,20R)-diol 8 (Fig. 1). To support the proposedrelative stereochemistry of the 19-alcohol and 26,27-stereo-centres, the analogous (2R,3R,20S)-diol was also synthesized and,as expected, the NMR data of this compound gave a poor t withthe natural product 3 (ESI, Tables 3 and 4, Fig. S2 and S3†).

With the above information in hand, it was necessary todetermine the conguration of the C-15 stereocenter as well asconrm the tentative assignment of absolute stereochemistry(17S, 19R, 26R, 27R) for diol 2. We turned to natural productdegradation and comparison with a synthetic standard (Scheme3). Ozonolysis of diol 2 followed by treatment with AcOH/H2O2

led to cleavage of the 12,13-double bond to give a dihydroxy acid

Fig. 1 1H NMR data comparison for synthetic fragments 8 and 9 with27-descarbamoyl metabolite 3.

This journal is © The Royal Society of Chemistry 2017

which cyclized, aer purication by HPLC, to give lactone 10 asa single diastereomer with [a]D +25.0.

From a combination of TOCSY and NOESY it was apparentthat the ring adopted a boat conformation with NOESY corre-lations from 5-H to 2a-H, 4a-H and 3-CH3 consistent with thesebeing on the same face of the molecule (Fig. 2). A NOESYcorrelation between 2b-H and 3-H, which was in accord with themethyl group at C-3 having a relative anti relationship with theC-5 side-chain so that in the ‘open chain structure’ it would havea syn relationship with the C-5 alcohol.

Further support came from comparing the couplingconstants for the ring protons in lactone 10 with those reportedfor anti and syn lactones 11 and 1222 (Fig. 2). The signalsassigned to 4-H2 in 11 and 12 have signicantly differentcouplings constants and the NMR data for anti lactone 11correlated well with those for degradation product 10.

The deduced structure and absolute conguration ofdegradation product 10 were conrmed by total synthesis(Schemes 4 and 5). Addition of a copper mediated Grignardreagent to chiral crotonate 13 using the conditions reported byLipshutz23 gave the known allylated sultam 14 generating the 3-methyl stereocenter in the target lactone. Oxidative cleavage ofthe alkene using OsO4/NaIO4 in the presence of 2,6-lutidine24

followed by reduction of the resultant aldehyde gave alcohol 15in quantitative yield. Addition of lutidine was important aswithout it only a 15% yield of aldehyde was obtained. Protectionof alcohol 15 as the TBS ether and reductive cleavage of theauxiliary gave an aldehyde which was immediately subjected toBrown allylation conditions with (+)-DIP-Cl25,26 giving alkene 16in 83% yield over the 3 steps. The stereochemical outcomeof the reaction was conrmed by analysis of the (R)- and(S)-Mosher's ester27,28 derivatives of alcohol 16.

Fig. 2 Selected NMR coupling constants for lactones 10, 11 and 12 andnOe correlations for 10.

Chem. Sci.

Scheme 4 Synthesis of amine 22.

Chemical Science Edge Article

Ope

n A

cces

s A

rtic

le. P

ublis

hed

on 1

1 Ju

ly 2

017.

Dow

nloa

ded

on 1

7/07

/201

7 14

:08:

41.

Thi

s ar

ticle

is li

cens

ed u

nder

a C

reat

ive

Com

mon

s A

ttrib

utio

n 3.

0 U

npor

ted

Lic

ence

.View Article Online

Next the amine and the further required protected alcoholwere introduced via manipulation of the terminal alkene of 16(Scheme 4). 1-Azido-2-trimethylsiloxanes have been prepared viakinetic resolution of terminal epoxides using TMSN3 and a chiralbidentate salen ligand.29 Thus alcohol 16 was protected as theTBS ether then treatment with MCPBA gave a 1.7 : 1 mixture ofepoxides 17 and 18 in 81% yield. The mixture was treated withchiral salen complex 19 and TMSN3 giving azide 20 (62%) andunreacted epoxide 18 (31%) which were readily separated bycolumn chromatography. The anti relationship of the protected1,3-diol was conrmed by deprotection of silyl ether 20 andconversion of the resultant anti 1,3-diol to acetonide 21 whichgave characteristic 13C NMR signals at dc 100 for the quaternary

Scheme 5 Preparation of lactone 10.

Chem. Sci.

carbon and dc 24.7, 24.8 for the methyl groups. TBS protection ofsecondary alcohol 20 and reduction of the azide gave amine 22required for coupling to the butanoic acid derivative.

Acid 23 was readily prepared from ethyl (2R,3R)-3-hydroxy-2-methylbutanoate30,31 via formation of the p-methoxybenzyl etherfollowed by hydrolysis of the ester with barium hydroxide(Scheme 5). Amine 22 and acid 23 were coupled giving 24 withthe required carbon framework of the target lactone. Furtherfunctional groupmanipulations included selective deprotectionof the primary silyl ether 24 then a two-step oxidation protocolto give carboxylic acid 25 using DMP followed by a Pinnickoxidation.32

Treatment of 25 with 1% HCl in EtOH generated lactone 26.To complete the synthesis of 10 the secondary alcohol in 26 wasprotected as the silyl ether and the benzyl ether removed with2,3-dichloro-5,6-dicyanobenzoquinone (DDQ) giving 27. Thecarbamoyl group was introduced using tri-chloroacetylisocyanate in CH2Cl2 followed by stirring withalumina to give 28 in 76% yield. Finally removal of the silyl etherwith 1% HCl in EtOH gave lactone 10.

The 1H- and 13C NMR data of synthetic lactone 10 and theproduct isolated from ozonolysis of metabolite 2 were the same(Table 5, ESI†). Furthermore the optical rotation of the twosamples were entirely consistent. Thus, we can conrm theabsolute and relative conguration of the C14–C28 fragment ofdiol 2 is as shown in Scheme 5.

The nal challenge was to determine the stereochemistry ofthe remote C-5 stereocentre. We again applied a combination ofdegradation studies and chemical synthesis. Diol 2 was meth-ylated with TMS diazomethane then treated with 2nd generationHoveyda–Grubbs catalyst33 under an ethylene atmosphere togive the degradation product (�)-29 with [a]D �6.8 (Scheme 6).

The absolute conguration of 29 was conrmed via theenantioselective synthesis shown in Scheme 7. The stereocentrewhich would become C-5 in the target was readily generated viaan alkylation reaction with NaHMDS/CH3I giving oxazolidinone30 as previously described.34 Following reductive cleavage of theauxiliary, primary alcohol 31 was converted to iodide 32.

The key synthetic step was formation of the tri-substitutedunsaturated ester 33. Preliminary studies using Horner–Wads-worth–Emmons or Julia chain extensions of a methyl ketoneproved unsatisfactory giving a mixture of E and Z-alkenes.

This journal is © The Royal Society of Chemistry 2017

Scheme 6 Methylation of diol 2 followed by degradation to ester(�)-29.

Scheme 7 Enantioselective synthesis of ester (+)-29 and finalassignment of diol 2.

Edge Article Chemical Science

Ope

n A

cces

s A

rtic

le. P

ublis

hed

on 1

1 Ju

ly 2

017.

Dow

nloa

ded

on 1

7/07

/201

7 14

:08:

41.

Thi

s ar

ticle

is li

cens

ed u

nder

a C

reat

ive

Com

mon

s A

ttrib

utio

n 3.

0 U

npor

ted

Lic

ence

.View Article Online

However, excellent control of alkene geometry was achieved viaaddition of the cuprate derived from iodide 32 to commerciallyavailable methyl but-2-ynoate. Following work up and depro-tection, primary alcohol 33 was isolated in 71% yield fromiodide 32 and the E-alkene geometry was conrmed by nOe.

Aldehyde 34, prepared by Dess–Martin periodinane(DMP) oxidation35 of alcohol 33, was coupled with but-3-enylmagnesium bromide and further oxidation gave ketone 35in 52% yield over the 3 steps. In the nal methylenation step itwas important to avoid bond migration of the exo alkene to anendo position. This was achieved via the zirconium-mediated

This journal is © The Royal Society of Chemistry 2017

methylenation36,37 of the ketone. Unsaturated methyl ester(+)-29 had identical NMR spectra to the degradation product(�)-29 but with an optical rotation [a]D +5.0 in accord with thesynthetic material being the enantiomer of the degradationproduct. Thus kalimantacin A 1 has the 5R conguration.

Conclusions

In conclusion, elucidating the absolute conguration ofcomplex polyketides with multiple stereogenic centers is oenan ambitious undertaking.38,39 Herein we used a multidisci-plinary approach involving natural product isolation, chemicaldegradation studies, bioinformatics, NMR correlations andfragment synthesis to rigorously determine the absolute andrelative stereochemistry of the kalimantacins isolated from P.uorescens. Diol 2 from the DbatM mutant has the 5R, 15S, 17S,19R, 26R, 27R stereochemistry and is the immediate biosyn-thetic precursor of the bioactive kalimantacin A 1 formed byoxidation of the secondary alcohol to the 17-ketone. The abso-lute congurations of the 17, 19 and 27-hydroxyl groups in diol2 are in good agreement with bioinformatic predictions.12,13

The 13C and 1H NMR data for natural product 1 isolated fromP. uorescens are entirely consistent with those reported4,5 forkalimantacin from Alcaligenes sp. YL-02632S in accord withboth structures having the same relative stereochemistry.Furthermore it is evident from comparison of PKS/NRPS geneclusters for kalimantacin A from cultures of P. uorescens12 andbatumin from P. batumici14 that the natural products are indeedthe same compound. Synergising chemical and biochemicalmethods have led to the elucidation of the structures of kali-mantacin A 1 (and hence batumin) and diol 2.

The inexorable rise of antibiotic resistant bacteria requiresa multi-facetted response to meet this global threat to humanhealth. In contrast to broad spectrum antibiotics, narrowspectrum agents can selectively decolonise specic pathogens(so called decolonisation drugs) in the human microbiota andare becoming increasingly important for detecting as well asghting specic infections. They may also reduce the chance ofemergence of antibiotic resistant strains.11 Kalimantacin hasbeen identied as a highly specic agent against staphylococcithat will potentially allow its deployment for the detection ofresistant strains as well as a potent decolonisation agent. Thedetermination of the absolute stereochemistry of kalimantacinas described in this paper will underpin further functionalinvestigation of this important molecule.

Acknowledgements

We thank Dr Kate de Mattos-Shipley for assistance with ourcomparison of gene clusters conrming literature. We aregrateful to the following for funding: the BBSRC and EPSRCincluding through BrisSynBio Synthetic Biology ResearchCentre (BB/L01386X/1) and the Bristol Chemical SynthesisCentre for Doctoral Training which provided PhD studentshipsfor IRGT (EP/G036764/1) and PDW (EP/L015366/1) and BaSe-icsresearch community from the FWO Vlaanderen (W.0014.12N).

Chem. Sci.

Chemical Science Edge Article

Ope

n A

cces

s A

rtic

le. P

ublis

hed

on 1

1 Ju

ly 2

017.

Dow

nloa

ded

on 1

7/07

/201

7 14

:08:

41.

Thi

s ar

ticle

is li

cens

ed u

nder

a C

reat

ive

Com

mon

s A

ttrib

utio

n 3.

0 U

npor

ted

Lic

ence

.View Article Online

Notes and references

1 K. J. Weissman and P. F. Leadlay, Nat. Rev. Microbiol., 2005,3, 925–936.

2 C. M. Thomas, J. Hothersall, C. L. Willis and T. J. Simpson,Nat. Rev. Microbiol., 2010, 8, 281–289.

3 C. Hertweck, Angew. Chem., Int. Ed., 2009, 48, 4688–4716.4 K. Kamigiri, Y. Suzuki, M. Shibazaki, M. Morioka, K. Suzuki,T. Tokunaga, B. Setiawan and R. M. Rantiatmodjo, J.Antibiot., 1996, 49, 136–139.

5 T. Tokunaga, K. Kamigiri, M. Orita, T. Nishikawa,M. Shimizu and H. Kaniwa, J. Antibiot., 1996, 49, 140–144.

6 V. V. Smirnov, L. N. Churkina, V. I. Perepnikhatka,N. S. Mukvich, A. D. Garagulia, E. A. Kiprianova,A. N. Kravets and S. A. Dovzhenko, Prikl. Biokhim.Mikrobiol., 2000, 36, 55–58.

7 V. V. Smirnov, L. N. Churkina, A. N. Kravets andV. I. Perepnikhatka, Appl. Biochem. Microbiol., 2000, 36,262–265.

8 L. N. Churkina, S. I. Bidnenko, G. Lopes dos Santos Santiago,M. Vaneechoutte, L. V. Avdeeva, O. B. Lutko andN. M. Oserjanskaja, BMC Res. Notes, 2012, 5, 374.

9 L. Churkina, M. Vaneechoutte, E. Kiprianova, N. Perunova,L. Avdeeva and O. Bukharin, Open J. Med. Microbiol., 2015,5, 193–201.

10 P.-Y. Levy, M. Ollivier, M. Drancourt, D. Raoult andJ.-N. Argenson, Orthopaedics & Traumatology: Surgery &Research, 2013, 99, 645–651.

11 E. Tacconelli, I. Autenrieth and A. Peschel, Science, 2017,355, 689–690.

12 W. Mattheus, L. J. Gao, P. Herdewijn, B. Landuyt,J. Verhaegen, J. Masschelein, G. Volckaert and R. Lavigne,Chem. Biol., 2010, 17, 149–159.

13 W. Mattheus, J. Masschelein, L. J. Gao, P. Herdewijn,B. Landuyt, G. Volckaert and R. Lavigne, Chem. Biol., 2010,17, 1067–1071.

14 V. V. Klochko, Biotechnol. Acta, 2014, 7, 46–50.15 V. V. Klochko, L. B. Zelena, J. Y. Kim, L. V. Avdeeva and

O. N. Reva, Int. J. Antimicrob. Agents, 2016, 47, 56–61.16 J. Piel, Nat. Prod. Rep., 2010, 27, 996–1047.17 E. J. N. Helfrich and J. Piel,Nat. Prod. Rep., 2016, 33, 231–316.

Chem. Sci.

18 V. E. Lee and A. J. O'Neill, Int. J. Antimicrob. Agents, 2017, 49,121–122.

19 D. A. Evans, K. T. Chapman and E. M. Carreira, J. Am. Chem.Soc., 1988, 110, 3560–3578.

20 K. Narasaka and F. C. Pai, Tetrahedron, 1984, 40, 2233–2238.21 S. D. Rychnovsky, B. N. Rogers and T. I. Richardson, Acc.

Chem. Res., 1998, 31, 9–17.22 G. Fronza, C. Fuganti, P. Grasselli and M. Terreni,

Tetrahedron, 1992, 48, 7363–7372.23 B. H. Lipshutz and C. Hackmann, J. Org. Chem., 1994, 59,

7437–7444.24 W. Yu, Y. Mei, Y. Kang, Z. Hua and Z. Jin, Org. Lett., 2004, 6,

3217–3219.25 H. C. Brown and P. K. Jadhav, J. Am. Chem. Soc., 1983, 105,

2092–2093.26 U. S. Racherla and H. C. Brown, J. Org. Chem., 1991, 56, 401–

404.27 J. A. Dale, D. L. Dull andH. S. Mosher, J. Org. Chem., 1969, 34,

2543–2549.28 J. A. Dale and H. S. Mosher, J. Am. Chem. Soc., 1973, 95, 512–

519.29 J. F. Larrow, S. E. Schaus and E. N. Jacobsen, J. Am. Chem.

Soc., 1996, 118, 7420–7421.30 K. Mori and T. Ebata, Tetrahedron, 1986, 42, 4413–4420.31 G. Frater, U. Muller and W. Gunther, Tetrahedron, 1984, 40,

1269–1277.32 B. S. Bal, W. E. Childers and H. W. Pinnick, Tetrahedron,

1981, 37, 2091–2096.33 S. B. Garber, J. S. Kingsbury, B. L. Gray and A. H. Hoveyda, J.

Am. Chem. Soc., 2000, 122, 8168–8179.34 R. Bajpai, F. Yang and D. P. Curran, Tetrahedron Lett., 2007,

48, 7965–7968.35 D. B. Dess and J. C. Martin, J. Org. Chem., 1983, 48, 4155–

4156.36 J. M. Tour, P. V. Bedworth and R.Wu, Tetrahedron Lett., 1989,

30, 3927–3930.37 M. Hartmann and E. Zbiral, Tetrahedron Lett., 1990, 31,

2875–2878.38 D. Menche, Nat. Prod. Rep., 2008, 25, 905–918.39 J. W. Blunt, B. R. Copp, R. A. Keyzers, M. H. G. Munro and

M. R. Prinsep, Nat. Prod. Rep., 2016, 33, 382–431.

This journal is © The Royal Society of Chemistry 2017